For each chromosome to properly segregate during mitosis, its kinetochores must bipolarly attach spindle microtubules. The failure of chromosomes to biorient is a major cause of cellular aneuploidy, a driving force in cancer and birth defects. Bipolar attachment is achieved because tension is produced between sister kinetochores, which both stabilizes microtubule attachments and turns off spindle checkpoint signals. A key to understanding how cells become aneuploid is to understand how chromosomes sense tension between sister kinetochores and use this to regulate microtubule attachment and spindle checkpoint signals. Proteins that localize to the inner centromere are central to these processes and these proteins form a network to regulate the Aurora B kinase which is a member of the chromosome passenger complex. We have purified the CPC to homogeneity and developed a system to study its activation in vitro. These experiments are uncovering both positive and negative feedback loops as well as the key mutants to dissect the role of these pathways in vivo. To characterize mutants we are employing the animal caps of Xenopus embryos which allow us to easily knockdown and replace proteins and dissect phenotypes in normal diploid tissue. The combination of in vitro biochemistry, Xenopus extracts and now dissection of phenotypes in animal caps provides a unique opportunity to move seamlessly between biochemical and cell biological approaches in a vertebrate system. We hypothesize that one role of the CPC is to generate gradients of soluble phosphoactivity that provide spatial information to pattern the 3D space of the cell for mitotic events. We will also test this important hypothesis as well as determine the role of Aurora B in generating a central band of RhoA that determines the location of the cytokinetic furrow. Finally we will perform purification of inner centromere chromatin to systematically identify proteins that localize to this chromosome territory as well as the DNA sequences that they are assembled upon.

Public Health Relevance

The missegregation of chromosomes during mitosis is a major source of genetic mutations in cancer. During mitosis every chromosome assembles an inner centromere between its kinetochores, which is a key signaling center to ensure accurate chromosome segregation. The experiments in this proposal systematically dissect the inner centromere region with an emphasis on the regulation of the Chromosome Passenger Complex, which includes the Aurora B kinase. The experiments employ the power of Xenopus extracts to dissect function and reconstitution of complex reagents from purified proteins. We also expand the Xenopus system by employing phenotypic characterization of cell cycle phenotypes in Xenopus embryos. This combination of biochemical, cell biological and in vivo techniques provides unique experimental power to dissect this important problem.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063045-13
Application #
8442871
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Hamlet, Michelle R
Project Start
2001-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
13
Fiscal Year
2013
Total Cost
$306,953
Indirect Cost
$106,329
Name
University of Virginia
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Trivedi, Prasad; Stukenberg, P Todd (2016) A Centromere-Signaling Network Underlies the Coordination among Mitotic Events. Trends Biochem Sci 41:160-74
Manukyan, Arkadi; Ludwig, Kirsten; Sanchez-Manchinelly, Sergio et al. (2015) A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells. J Cell Sci 128:50-60
Eagleson, Gerald; Pfister, Katherine; Knowlton, Anne L et al. (2015) Kif2a depletion generates chromosome segregation and pole coalescence defects in animal caps and inhibits gastrulation of the Xenopus embryo. Mol Biol Cell 26:924-37
Stukenberg, P Todd; Burke, Daniel J (2015) Connecting the microtubule attachment status of each kinetochore to cell cycle arrest through the spindle assembly checkpoint. Chromosoma 124:463-80
Zyłkiewicz, Eliza; Stukenberg, P Todd (2014) Xenopus egg extracts as a simplified model system for structure-function studies of dynein regulators. Methods Mol Biol 1136:117-33
Banerjee, Budhaditya; Kestner, Cortney A; Stukenberg, P Todd (2014) EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B. J Cell Biol 204:947-63
Matson, Daniel R; Stukenberg, P Todd (2014) CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status. J Cell Biol 205:541-54
Earnshaw, W C; Allshire, R C; Black, B E et al. (2013) Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21:101-6
Niedzialkowska, Ewa; Wang, Fangwei; Porebski, Przemyslaw J et al. (2012) Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres. Mol Biol Cell 23:1457-66
Alushin, Gregory M; Musinipally, Vivek; Matson, Daniel et al. (2012) Multimodal microtubule binding by the Ndc80 kinetochore complex. Nat Struct Mol Biol 19:1161-7

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